View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Covenant University Repository

Biotechnology Advances 29 (2011) 210–222

Contents lists available at ScienceDirect

Biotechnology Advances

journal homepage: www.elsevier.com/locate/biotechadv

Research review paper Advances in plant molecular farming

Olawole O. Obembe a,⁎, Jacob O. Popoola a, Sadhu Leelavathi b, Siva V. Reddy b

a Department of Biological Sciences, Covenant University, PMB 1023 Ota, Ogun State, Nigeria b Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India

article info abstract

Article history: Plant molecular farming (PMF) is a new branch of plant biotechnology, where plants are engineered to Received 27 July 2010 produce recombinant pharmaceutical and industrial proteins in large quantities. As an emerging subdivision Received in revised form 12 November 2010 of the biopharmaceutical industry, PMF is still trying to gain comparable social acceptance as the already Accepted 12 November 2010 established production systems that produce these high valued proteins in microbial, yeast, or mammalian Available online 27 November 2010 expression systems. This article reviews the various cost-effective technologies and strategies, which are being developed to improve yield and quality of the plant-derived pharmaceuticals, thereby making plant- Keywords: Plant-derived proteins based production system suitable alternatives to the existing systems. It also attempts to overview the Clinical development different novel plant-derived pharmaceuticals and non-pharmaceutical protein products that are at various Commercialization stages of clinical development or commercialization. It then discusses the biosafety and regulatory issues, Biosafety which are crucial (if strictly adhered to) to eliminating potential health and environmental risks, which in Regulation turn is necessary to earning favorable public perception, thus ensuring the success of the industry. © 2010 Elsevier Inc. All rights reserved.

Contents

1. Introduction ...... 211 2. Plant transformation strategies ...... 211 2.1. Stable nuclear transformation...... 211 2.2. Stable plastid transformation ...... 211 2.3. Plant cell-suspension cultures...... 211 2.4. Transient expression systems ...... 212 2.4.1. Agroinfiltration method ...... 212 2.4.2. Virus infection method ...... 212 2.4.3. The magnifection technology...... 212 3. Limitations and optimization of plant production systems...... 212 3.1. The problem of low yield...... 212 3.1.1. Optimizing transcript expression ...... 212 3.1.2. Optimizing protein's stability...... 213 3.2. The glycosylation challenge...... 213 3.3. Choice of suitable host plants ...... 213 3.3.1. Cost of downstream processing ...... 214 4. Overview of plant-derived recombinant proteins ...... 215 4.1. Plant-derived vaccine antigens ...... 215 4.2. Plant-derived antibodies ...... 215 4.3. Therapeutic and nutraceutical proteins ...... 217 4.4. Non-pharmaceutical plant-derived proteins ...... 217 5. Biosafety and regulatory issues ...... 217 5.1. Problem of co-mingling and contamination of the food/feed chain ...... 217 5.2. The problem of gene spread and unintended exposures ...... 218 5.3. Risk of horizontal gene transfer ...... 218 6. Concluding remarks...... 218

⁎ Corresponding author. Tel.: +234 8130928965. E-mail address: [email protected] (O.O. Obembe).

0734-9750/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2010.11.004 O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222 211

Acknowledgement ...... 218 References ...... 218

1. Introduction recombinant proteins till date. The method has been used to accumulate protein in the dry seeds of cereals, which allows long- Plant molecular farming (PMF) refers to the production of term storage of the seed at room temperature without degradation of recombinant proteins (including pharmaceuticals and industrial the protein (Horn et al., 2004). Additionally, the system has high proteins) and other secondary metabolites, in plants. This involves scale-up capacity, as crops such as the cereals are grown practically the growing, harvesting, transport, storage, and downstream proces- everywhere. However, a long production cycle of some crops and the sing of extraction and purification of the protein (Wilde et al., 2002). potential to cross with native species or food crops are limiting public This technology hinges on the genetic transformability of plants, acceptance of this method (Commandeur et al., 2003). which was first demonstrated in the 1980s (Bevan et al., 1983). The first recombinant plant-derived pharmaceutical protein (the human 2.2. Stable plastid transformation growth hormone) and the first recombinant antibody (expressed in the progeny of the cross of two individual transgenic plants Plastid transformation provides a valuable alternative to nuclear expressing single immunoglobulin gamma and kappa chains) were transformation because it combines numerous advantages that the produced in transgenic plants in 1986 and 1989, respectively (Hiatt nuclear transformation lacks, including the provision of a natural et al., 1989; Barta et al., 1986). However, it was not until 1997 that the biocontainment of transgene flow by out-crossing (as plastids are first recombinant protein, avidin (an egg protein) was expressed in inherited through maternal tissues in most species and the pollen transgenic maize for commercial purpose (Hood et al., 1997). These does not contain chloroplasts, hence the transgene may not be exploits really demonstrated that plants could be turned into bio- transferable, thereby allaying public concerns (Cardi et al., 2010; factories for the large scale production of recombinant proteins. It has Meyers et al., 2010). Transgenic plants with homoplastomic chloro- been proven over the years that plants have the ability to produce plast transformation (in which every chloroplast carries the trans- even more complex functional mammalian proteins with therapeutic gene) are selected after several generations of plant regeneration activity, such as human serum proteins and growth regulators, from bombarded leaf explants, on selection medium containing antibodies, vaccines, hormones, cytokines, enzymes and antibodies spectinomycin or in combination with streptomycin. Our group (Liénard et al., 2007). This has been possible due to their ability to (Reddy et al., 2002; Sadhu and Reddy, 2003) had previously achieved perform post-translational modifications that make these recombi- accumulation of a bacterial- and a human therapeutic protein, up to nant proteins fold properly and maintain their structural and 3–6% of total soluble proteins in tobacco chloroplasts. Recently, Oey functional integrity. et al. (2009) reported a whopping expression level (70% of the total The fast-growing market for recombinant biopharmaceuticals, soluble proteins) for a proteinaceous antibiotic, with the chloroplast which accounted for about 10% of the pharmaceutical market in 2007 system, which represents the highest recombinant protein accumu- (Lowe and Jones, 2007), had been predicted to expand to US $ lation achieved so far in plants. This enormous potentials notwith- 100 billion in 2010 (Knäblein, 2005). With increasing demand for bio- standing, plastid transformation is still limited in its applications, in pharmaceuticals, coupled with the high costs and inefficiency of the that even though it has been achieved in other plant species such as existing production systems (Knäblein, 2005), which include yeast, tomato, eggplant, lettuce, and soybeans (Bock, 2007; Singh and microbes, animals cells (Jones et al., 2003) and transgenic animals Verma, 2010), routine chloroplast transformation is only possible in (Harvey et al., 2002), there now exists a shortage in manufacturing tobacco, which is inedible and highly regulated, being rich in toxic capacity, to such an extent that some patients have to wait for these alkaloids. It is also envisaged that protein stability will change over products. Hence transgenic plants are gaining more attention as the time even with refrigeration (Horn et al., 2004). new generation bio-reactors; with active research and development activity in PMF identified in over one hundred and twenty companies, universities, and research institutes, around the world (Basaran and 2.3. Plant cell-suspension cultures Rodríguez-Cerezo, 2008). The weightier advantages of the plant- based system over the existing systems are the incentives for this. This Plant cell-suspension culture is an alternative plant-based pro- review also discusses the different technologies and the limitations of duction platform to mammalian cells for production of biopharma- the plant system with respect to the various approaches being ceutical. The system guarantees sterile in vitro conditions, coupled pursued to overcome the challenges in other to increase its social with high-level containment (Franconi et al., 2010)(idealfor acceptability. It also attempts to give an overview of the plant-derived production of high purity pharmaceuticals) and cheaper and simpler proteins and discusses the biosafety and regulatory issues of these downstream processing and purification (Kim et al., 2008). Addition- emerging technologies. ally, the use of suspension cultured cells as expression system could reduce heterogeneity of the protein and of the N-glycan, owing to the uniformity in the size and type of cells (Liénard et al., 2007; De 2. Plant transformation strategies Muynck et al., 2009). Moreover, the system is rapid, because there is no need to regenerate and characterize transgenic plants, such that 2.1. Stable nuclear transformation productive cell lines can be generated within a few months (Shaaltiel et al., 2007; Aviezer et al., 2009 ). Examples of plant suspension Stable nuclear transformation involves the incorporation of a culture-produced biopharmaceuticals include Dow Agroscience's foreign gene or genes (exogenous) of interest into the nuclear purified injectable Newcastle disease virus (NDV) vaccine for genome of the plant, thereby altering its genetic makeup, and leading chickens, which has been approved by the USDA Center for Veterinary to the expression of the transgene after integration with the host Biologics (http://www.dowagro.com/animalhealth) and Protalix's genome, thereby conferring stably inheritable traits that were not recombinant human glucocerebrosidase for the treatment of Gaucher's present in the untransformed host plant. As the most common disease (http://www.protalix.com, Shaaltiel et al., 2007). Though method, stable nuclear transformation has produced most of the cheaper, safer, easier to manipulate and more rapid than most 212 O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222 established conventional systems, suspension culture is still not the best wider host range, speed, safety, low cost and high quality and yield of production platform the plant system does offer, as the overall yield and pharmaceutical proteins required for clinical development (http:// usability is somewhat limited by the diminishing level of recombinant www.kbpllc.com, Komarova et al., 2010; Pogue et al., 2010; Rybicki, protein during the late stationary phase due to increased proteolytic 2009). activity (Corrado and Karali, 2009). Besides, the system is still limited to small number of well-characterized plant cell lines (such as tobacco, rice 3. Limitations and optimization of plant production systems carrot, or Arabidopsis)(Breyer et al., 2009). In spite of the vast advantages of the plant production systems 2.4. Transient expression systems over the established systems (Buonaguro and Butler-Ransohoff, 2010; Obembe, 2010), there exist however, some limitations that make The transient production platform is perhaps the fastest and the them have comparatively lower social acceptance. The problem of low most convenient production platform for plant molecular farming yield (which can result from low-level transcript expression or the (Rybicki, 2010). The systems, which are mainly used for quick instability of the recombinant protein) and non-mammalian glyco- validation of expression constructs, are now being used routinely for sylation are the two major challenges that militate against the full the production of considerable amounts of proteins within a few exploitation of plants as alternative bioreactors to the mammalian cell weeks (Vézina et al., 2009). These include the following methods. culture, especially. The challenge of choosing the most suitable host plant for any specific biomolecule as well as downstream processing is 2.4.1. Agroinfiltration method also being given due attention. The agroinfiltration method, which was developed by Kapila et al. (1997), involves infiltration of a suspension of recombinant Agrobac- 3.1. The problem of low yield terium tumefaciens into tobacco leaf tissue, which facilitates the transfer of T-DNA to a very high percentage of the cells, where it 3.1.1. Optimizing transcript expression expresses the transgene at very high levels without stable transfor- In order to optimize transcript expression, the general strategy is mation, as in the case of transgenic crops. This method has now been the use strong and constitutive promoters, such as the cauliflower developed into a very rapid, high-yielding transient expression mosaic virus 35S RNA promoter (CaMV 35S) and maize ubiquitin-1 strategy for producing clinical grade bio-pharmaceuticals (Vézina promoter (ubi-1), for dicots and monocot, respectively (Fischer et al., et al., 2009; Pogue et al., 2010; Regnard et al., 2010). 2004). Organ- and tissue-specific promoters are also being used to drive expression of the transgenes (vaccine antigen HBsAg M, murine 2.4.2. Virus infection method single chain variable fragment (scFv) G4 and human interferon-α)in The virus infection method is dependent on the ability of plant the tissue or organ like the tuber, the seed and the fruit (He et al., viruses such as tobacco mosaic virus (TMV) and potato virus X (PVX) 2008; De Jaeger et al., 2002; Masumura et al., 2006). This tissue- to be used as vectors to deliver foreign genes into plants, without specific expression therefore prevents accumulation of the recombi- integration (Porta and Lomonossoff, 2002). Both expression platforms nant protein in the vegetative organs, which might negatively affect infect tobacco plants and then transiently express a target protein in plant growth and development. Additionally, inducible promoters, the plant tissue. Meanwhile, Varsani et al. (2006) were able to whose activities are regulated by either chemical or external stimulus, successfully obtain protein yield as high as 17% of the total protein, may equally be used to prevent the lethality problem (Corrado and using this expression method. However, as with other fresh plant- Karali, 2009), as it is being used in cell suspension cultures (Nara et al., based production systems, the recombinant protein has to be 2000; Peebles et al., 2007). Besides, transcription factors can be used processed immediately to prevent tissue degradation and of course as boosters for the promoters to further enhance the expression level protein instability. This represents the only minor drawback of this of the transgenes (Yang et al., 2001). Moreover, it has been recently system. Nonetheless, a pharmaceutical company, Large Scale Biology found that the terminator of the heat-shock gene of Arabidopsis Corporation has adopted the system for the production of idiotype thaliana shows an increase in transcription of a foreign gene by four vaccines for the treatment of B-cell non-Hodgkin's lymphoma, which times (Nagaya et al., 2010). have successfully passed the phase I clinical testing (McCormick et al., Furthermore, the expression constructs can also be designed to 2008). ensure transcript stability and translational efficiency. This involves, for instance, the removal of the native 5′ and 3′ untranslated regions 2.4.3. The magnifection technology from the foreign gene and introducing 5′ untranslated leader Both the viral vector-based expression system and Agrobacterium- sequence of the tobacco mosaic virus RNA, rice polyubiquitin gene mediated method are limited in their inability to achieve high-level RUB13, alfalfa mosaic virus or tobacco etch virus in the expression co-expression of two or several polypeptides necessary for the construct, all of which have been separately shown to significantly assembly of heterooligomeric proteins (Giritch et al., 2006). Hence enhance the expression levels of transgenes (Lu et al., 2008; Sharma the evolution of a new robust transient expression system known as et al., 2008). In addition to these leader sequences, the expression MagnICON®technology, which was developed by Icon Genetics, by cassette design can be such that the false AU-rich sequences in the 3′ removing the coat protein (responsible for systemic movement) of untranslated regions that may act as splice sites are removed or the noncompeting virus strains and the systemic delivery of the modified, to ensure transcript stability (Mishra et al., 2006). Besides, resulting viral vectors to the entire plant using Agrobacterium as the the transcript stability can be ensured by co-expressing the gene of vehicle of delivery and primary infection (Gleba et al., 2005). This interest and a suppressor of RNA silencing (Voinnet et al., 2003). It is method has not only improved infectivity but also remarkably also established that each organism exhibits biased codon usage, such enhanced amplification and ultimately lead to high-level co-expres- that it might be important to adapt the coding sequence of the sion of several polypeptides, which were able to assemble functional heterologous gene to that of the host plant in order to optimize heterooligomeric proteins at much increased levels up to 100-fold, translation efficiency (Liénard et al., 2007; ). In this regard, the including a full sized IgG (Giritch et al., 2006) and Yersinia pestis translational start-site of the heterologous protein is modified to match antigens fusion protein F1–V(Rosales-Mendoza et al., 2010). with the Kozak consensus for plants (Kawaguchi and Bailey-Serres, Other advanced viral vectors are currently being designed by 2002) or by using the sequence GCT TCC TCC after initiation codon, or various platforms such as, Icon Genetics, Kentucky BioProcessing, ACC or ACA before it (Sharma and Sharma, 2009). The codon Fraunhofer, and host of others, to facilitate ease of manipulation, modification, however, should be empirically determined rather than O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222 213 predicted because of the variation in the levels of transgene expression region of a tail anchored (TA) protein (Maggio et al., 2007). Other in the same system, using the same construct (Rybicki, 2009). To this subcellular compartments like the protein-storing vacuoles are also end, codon combinations (A/G)(a/c)(a/g) AUG and (A/G)(u/C)(g/C) now being explored for accumulating recombinant protein, as it has AUG have been reported to be optimum for enhanced translational been observed in rice seed endosperm (Yang et al., 2003). Extracel- activity in Arabidopsis and rice, respectively (Sugio et al., 2010). This lular proteolytic cleavage is another important factor for consider- variation in the expression of the transgene may be due to position ation in plant-based production of pharmaceuticals (Komarnytsky effect, copy number of the transgene or gene silencing. With respect et al., 2006; De Muynck et al., 2009). Komarnytsky et al. (2006) were to position effect, expression cassettes can now be designed to able to achieve a threefold increase in expression of an antibody when contain the nuclear matrix attachment regions (MAR), which are co-expressed with a secreted form of the Bowman–Birk serine regulatory sequences that ensure the placement of the transgene in peptidase inhibitor than when co-expressed with a cytosolic form of suitable regions for mobilizing transcription factors to the promoters this inhibitor. The addition of gelatin as a substitution substrate for (Streatfield, 2007). Besides, the problem of position effect can be peptidases was also found to increase antibody levels to as high as avoided by targeting the transgene into the plastids (Cardi et al., 300% in tobacco rhizosecretion (Drake et al., 2003). In addition, 2010). For optimizing the generation of single-copy transgenics, the suspension cells culture media formulations that are rich in peptidase strategies that have been used include the use of specific genetic inhibitor might provide safe in vitro environments for human IgG, elements, including the cAMP response elements (CREs), for co- especially in tobacco. Better still, it would be worthwhile to develop transfer with transgene in the T-DNA (De Paepe et al., 2009). extracellular peptidase-free host plants or to engineer antibodies Additionally, a new technology, which consists in the construction of displaying higher resistance to peptidases while maintaining their genetically autonomous artificial minichromosomes has been de- activity (De Muynck et al., 2010). scribed as providing infinite possibilities with several enormous advantages including gene stability; owing to the absence of gene 3.2. The glycosylation challenge silencing and position effect (Ananiev et al., 2009). Glycosylation is the covalent linkage of sugar moieties to proteins, 3.1.2. Optimizing protein's stability in order to enhance their folding, biological activity, solubility and For optimizing the stability of recombinant protein, which has bioavailability (Liénard et al., 2007). In plant, glycosylation occurs in been considered as the single most important factor limiting the the secretory pathway in the ER and the Golgi apparatus. However, yields in molecular farming (Schillberg et al., 2005) certain subcellular there are differences in the glycosylation patterns between the plants targeting might be needed. Not only does subcellular targeting and animals, with respect to N-glycan composition. Plants add α(1,3) enhance protein stability, but it also determines the type of the fucose and β(1,2) xylose residues to the N-glycan of their glycopro- associated protein processing, and by adding fusions and affinity tags, teins, whereas mammals add α(1,6) fucose moieties, glucose and it can be used to improve downstream processing of isolation and sialic acid residues to the N-glycan. Hence, to prevent the problem of purification (Fischer et al., 2004). Proteins can be targeted to the immunogenicity and allergic reactions that these differences could secretory pathway by an N-terminal signal peptide, which is cleaved potentially cause, when plant-derived therapeutic animal proteins are off for the release of the protein into the endoplasmic reticulum (ER). administered to humans (Krapp et al., 2003), there is need to engineer Nonetheless, full-size immunoglobulins that are addressed to the the plant to perform authentic human N-glycosylation. There are a secretory pathway have been shown to accumulate at higher levels number of strategies that have been used to modify the N- and easily recovered when fused with to a stabilizing partner glycosylation pattern in the plants (Gomord et al., 2010). These (Benchabane et al., 2008; Floss et al., 2009). Most plant-derived include the fusion of the ER-retention signal KDEL, which restricted recombinant proteins produced so far have been secreted into the glycosylation of plant-derived antibodies to only high mannose-type apoplastic space of the cell wall (Streatfield, 2006), and have been N-glycan (Floss et al., 2009), even though, however, such antibodies reported to accumulate at levels up to 100-fold greater than in the with high mannose N-glycan have been reported previously to be cytosol (Schillberg et al., 1999). Recent evidence however suggests unstable after injection into mouse (Mainieri et al., 2004). Other that the tomato cathepsin D inhibitor (SlCDI) has the potential to be strategies that have been used include generation of knock-out used as an in-built protein-stabilizing agent for the protection of mutant lines in Arabidopsis and a moss plant, Physcomitrella patens, cytosol-targeted recombinant proteins in plants (Goulet et al., 2010). that synthesize complex N-glycans lacking immunogenic xylose and The stability and accumulation of recombinant protein could be fucose epitope (Koprivova et al., 2004; Kang et al., 2008), and further increased by targeting to and retaining in other subcellular modulation of the xylosylated and fucosylated glycan levels, using compartments of the secretory pathways, such as the ER (Conrad and RNA-interference (Sourrouille et al., 2008), as well as co-expression of Fiedler, 1998). This is accomplished by appending of SEKDEL ER an RNA-interference construct with the therapeutic protein (Cox et al., retention signal to the C-terminus of the protein (Rademacher et al., 2006). Also, the knock-in strategy, which involves the expression of a 2008) or by using the N- or C-terminus fusion of γ-zein signal, which hybrid enzyme obtained through domain fusion of the CST domain of has been found to be more efficient and more economical for human β-1,4-galactosyltransferase I with β-1,2-xylosyltransferase of production of recombinant proteins (Mainieri et al., 2004; Torrent Arabidopsis, was shown to cause high-level galactosylation of N- et al., 2009a, 2009b). glycans and sharp decrease in the level of N-glycans with xylose and Proteins that do not require post-translational modification like fucose moieties (Vézina et al., 2009). Moreover, synthesis of sialic acid glycosylation for their activity can be targeted to the chloroplast, or in plant and sialylation of plant-expressed protein has also been better still the foreign gene can be used to transform chloroplast reported (Castilho et al., 2010). Hence, it is clear that glycosylation directly, with highly enhanced protein accumulation. Moreover, post- engineering is effective in adapting plant-made antibodies (De translational modifications of the ER lumen can also be avoided by Muynck et al., 2010), thus expanding the possibilities for the expressing the protein as translational fusion with oleosin protein, production of optimally glycosylated proteins with enhanced biolog- which target the expression of the foreign protein to oil bodies of the ical activities for use as human therapeutics (Loos et al., 2010). seeds (Moloney and Siloto, 2004), which in addition facilitates easy purification of the protein (as discussed later under cost of 3.3. Choice of suitable host plants downstream processing). Besides, proteins can be targeted to membrane surfaces and protected from cytosolic degradation without The choice of suitable host for deploying the molecular farming necessarily passing through the ER lumen by fusing the C-terminal technology is crucial for its overall success. In fact, though subtle, this 214 O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222 constitutes the first line of strategy for ensuring efficiency of having the highest biomass yield among food crops, ease of recombinant protein production. Hence, economic consideration is transformation, and ease of scale-up (Ramessar et al., 2008a), which the key issue in choosing suitable host plants for molecular farming. explains why Prodigene Inc. preferred to use maize for the The important economic factors include total biomass yield, storage commercial production of the first recombinant protein, avidin properties (facilities), ease of transportation, value of the recombinant (Hood et al., 1997) and bovine trypsin (Woodard et al., 2003). The protein itself, set-up costs, scale-up and maintenance costs, costs of Pharma-Planta Consortium has also adopted it for the production of a containment, availability of labor, land area required, length of HIV microbicide (Ramessar et al., 2008b). In addition to sharing many production cycle, costs of downstream processing and edibility of the advantages of maize, rice is a self-pollinating plant, as such it (Fischer et al., 2004; Schillberg et al., 2005). The suitable host should, would have a lower risk of unintended gene flow, which is why in addition to economic consideration, be amenable to transformation Ventria Bioscience has pioneered the production of two rice-derived and regeneration. Hence, the most suitable host plant for a particular proteins, human lactoferrin and lysozyme, which have received recombinant protein has to be determined empirically, based on these regulatory approvals, and have already been marketed (http:// highlighted factors, as there is no single perfect system thus far www.ventria.com, Boothe et al., 2010; Huang et al., 2008; Lau and (Schillberg et al., 2005). Sun, 2009). Similarly, ORF genetics Inc. is now exploiting the great In addition to being highly amenable to transformation and potential of cereal seeds (barley) in producing their two commercial regeneration, tobacco also combines most of these economic products ISOkine™ and DERMOkine™ human-like growth factors, advantages, such as high biomass yield, high scale-up capacity, non- thereby bypassing the use of bacterial or animal cell systems (http:// food/non-feed status (providing containment of transgenic material www.orfgenetics.com/, Faye and Gomord, 2010). The advantage of from contaminating food and feed chain), well-established transfor- exceptionally high protein content (20–40%) in legume seeds, mation protocols, all-year round growth and harvesting, and coupled with the self-pollinating status of soybean and pea has availability of large scale infrastructure for processing (Biemelt and made them candidate platform for accumulating recombinant Sonnewald, 2005; Fischer et al., 2004; Stoger et al., 2005a), which proteins. Soybean, in particular, has been explored for its efficacy to explains why it is most preferred for molecular farming of express a humanized antibody against herpes simplex virus, bovine pharmaceutically relevant proteins including antibodies, vaccines casein, and a human growth hormone (Philip et al., 2001; Russell and immunomodulatory molecules such as cytokines (De Muynck et al., 2005; Zeitlin et al., 2009). Soybean was also used to express a et al., 2010; Tremblay et al., 2010); many of which are at various functional hypotensive peptide, which reduced the systolic blood stages of development and even commercialization, as in the case of pressures of model mice (Yamada et al., 2008). CaroRX™, which is being used for preventing tooth decay (Ma et al., 1998). However, tobacco (except cultivar “81V9, Menassa et al., 3.3.1. Cost of downstream processing 2001), contains high amounts of toxic compounds, nicotine and other The cost of downstream processing of plant-derived recombinant alkaloids which have to be removed during purification process. proteins is believed to account for about 80% of the total production Besides, finding a beneficial use for tobacco in place of smoking costs (Buonaguro and Butler-Ransohoff, 2010; Kusnadi et al., 1998); (which has been banned in public places in most of the developed as such it has been given its well-deserved attention with respect to countries and nowadays in some developing countries) and providing devising strategies to reduce it to the barest minimum. However, an alternative source of income for tobacco farmers through PMF do using watery tissues such as tomatoes as the production systems has not seem to appeal to the existing negative public sentiment against been advanced to have the potential of lowering this cost, as it is tobacco in the United Kingdom, for instance (Milne, 2008). However, easier to extract from them than dry tissue like cereals (Schillberg the negative perception of tobacco with respect to its beneficial use et al., 2005; Yano et al., 2010). Additionally, with respect to biosafety might not be an issue, especially when the PMF proteins are not for concern on containment of the transgenic plants, tomatoes also human consumptions (e.g. for topical applications) and their appear attractive as a host crop, because they are grown in production are carried out in closed containment facilities, as such greenhouses. Nowadays, the oil bodies in certain oilseed crops like tobacco-based production might still maintain its relevance for a long safflower and rape seeds are now being exploited to ease recombinant time to come, just as the public comes to term with the fact that this protein purification, and minimizing the downstream costs, by using plant can as well be an indirect life-saver. Nevertheless, in the the oleosin fusion technology developed by SemBioSys Genetics meantime, alternative leafy crops like alfalfa and lettuce are now (http://www.sembiosys.com/). The strategy involves targeting the being explored as suitable hosts for molecular farming (Rosales- recombinant protein to the seed of the oilseed crops as a fusion with Mendoza et al., 2010). Leafy crops, however, generally have a oleosin, hence simplifying the extraction of the fusion proteins from common problem of instability of the expressed proteins, which the oil bodies and the release of the recombinant protein from its necessitate that leaves have to be desiccated, or frozen or processed fusion partner, for example in the case of accumulating and purifying immediately after harvest (Fischer et al., 2004). Additionally, biologically active human insulin, Apolipoprotein AI (Milano) and expression in vegetative organs, such as the leaf could affect growth human growth hormone in safflower (Boothe et al., 2010; Nykiforuk and development of the particular plant. et al., 2006; Nykiforuk et al., 2010). Seed-based expression seems to be more ideal in this respect, in It is envisaged that the advantage of lower cost of production that, not only does it not affect the growth and development of the might facilitate the participation of less developed countries in plant but it does not need to be frozen or dried or processed pharmaceutical production in plants (Paul and Ma, 2010). There are immediately, as accumulation of protein in the seed offers protection indeed several plant-derived recombinant proteins that were earlier against proteolytic digestion, as such it allows long-term storage at thought to be delivered as edible vaccines, which can be eaten directly ambient temperature without the protein losing its activity (Stoger as fruits (tomatoes, banana) and vegetables (lettuce, carrot), thus et al., 2002; Nochi et al., 2007). Additionally, seed-based expression eliminating the need for any processing, hence no processing costs platforms are most competitive in applications that require large (Mason et al., 2002). Banana in particular, was being touted as a volume of recombinant proteins per annum (Faye and Gomord, candidate host fruit crop for producing edible vaccine, especially for 2010). Moreover, the seed-based system allows oral delivery of the developing countries, as there would not be need for long- vaccine antigens or pharmaceutical proteins for immunization, distance transport and satisfying cooling requirements, since it is immunotherapy, and treatment of diseases (Lamphear et al., 2002; widely grown in these countries (Biemelt and Sonnewald, 2005). Takagi et al., 2005). In this regard, the cereal seeds especially rice and Additional advantages of banana are that of high digestibility and maize are gaining prominence. Maize has many advantages such as palatability, as such they were earlier gaining public acceptance for O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222 215 infant vaccination (Kumar et al., 2004, 2005). The suitability of the potential for large scale production, production costs reduction potatoes for oral vaccine production had also generated interest and oral delivery option (Streatfield and Howard, 2003; Pujol et al., because they can be eaten raw or with just little processing. Potatoes, 2005; Buonaguro and Butler-Ransohoff, 2010) as well as being like seeds, have the advantage of product stability due to the potential hosts for the production of glycosylated subunit vaccines specialized molecular environment in the tuber (Sparrow et al., (Bosch and Schots, 2010). Several vaccines have been expressed in 2007). Several clinical trials of potato tuber-derived antigens have plants, since the first plant-derived vaccine-relevant protein was been successful (Streatfield and Howard, 2003; Walmsley and reported 20 years ago (Rybicki (2009; Tiwari et al., 2009). These Arntzen, 2003), however, for the crop to be acceptable for use in include the hepatitis B surface antigen, which has been expressed in oral vaccine administration, there must be some degree of processing, transgenic potatoes (Richter et al., 2000), in tomato (He et al., 2008), cooking, in order to destroy the endotoxins; a process that will in banana (Kumar et al., 2005) and in tobacco cell suspension culture degrade the thermolabile protein products (Sparrow et al., 2007). Due (Sojikul et al., 2003), the heat labile enterotoxin B subunit (LTB) of to ethical issues however, the consequential shift in research focus Escherichia coli, has ben expressed in potato tubers (Lauterslager et al., toward the development of heat stable oral vaccines (Rybicki, 2009)is 2001), in maize seed (Chikwamba et al., 2002), in tobacco (Rosales- now giving way to the production of plant-derived vaccines that Mendoza et al., 2009), and in soybean (Moravec et al., 2007). would still be processed further, formulated in a reproducible way The cholera toxin B subunit (CTB) of Vibrio cholera has been and given under supervision so as to ensure reproducible effects expressed in several crops, including tobacco, tomato and rice (Daniell (Rybicki, 2010). et al., 2001a; Mishra et al., 2006; Nochi et al., 2007), several plant- As a result of the demand for high containment of transgenic made vaccines for veterinary purposes including, avian influenza, plants these days, safer and more cost-effective alternative plant- Newcastle disease, foot-and-mouth disease and enterotoxigenic based expression platforms are now being sought after, including Escherichia coli have been expressed in the plant (Lentz et al., 2010; Physcomitrella patens, a moss and green algae, such as Chlamydomonas Ling et al., 2010), and the Hemagglutinin protein of rinderpest has reinhardtii. These two expression systems combine the intrinsic been expressed in pigeon pea and peanut (Satyavathi et al., 2003). advantage of being amenable to cultivation in bioreactors under Others include the L1 protein of human papillomavirus types 11 and controlled conditions (Decker and Reski, 2004, 2008; Franklin and 16 (Biemelt et al., 2003; Maclean et al., 2007; Giorgi et al., 2010), the Mayfield, 2004; Hohe et al., 2002; Tran et al., 2009) with the Norwalk virus capsid protein (Mason et al., 1996), the Hemagglutinin possibility of secreting of recombinant proteins into the medium protein from measles virus (Marquet-Blouin et al., 2003) and the (thus reducing the extraction and purification costs) (Mayfield and H5N1 pandemic vaccine candidate (D'Aoust et al., 2010), all of which Franklin, 2005), and with a simple transformation procedure and have been expressed in one or two of the following plants: tobacco, shorter production cycle (Leon-Banares et al., 2004; Schaefer, 2002). potato and carrots. Also worthy of note, is the US Defense Physcomitrella has even been engineered to produce a strain that does Department-sponsored research and development of various bio- not add immunogenic β(1,2) xylose and α(1,3) fucose (Koprivova defense vaccines against anthrax, ricin, plague and haemorrhagic et al., 2004), thus eliminating the concern about non-human fever virus (Hull et al., 2005; Koya et al., 2005; Santi et al., 2006; Mett glycosylation, which is the reason for its adoption by German biotech et al., 2007). company, Greenovation Biotech as their production platform, and Plant-produced antigens have been reported to induce immune uniquely branded as bryotechnology (http://www.greenovation. responses, conferring protection against challenge in mouse model com). Other example of plant-based expression system is the Lemna systems (Satyavathi et al., 2003; Streatfield and Howard, 2003). Oral plant, which has very high rate of biomass accumulation per unit administration of several plant-produced antigens has been reported of time; doubling in size every 24–48 h (Liénard et al., 2007) and to induce protective immune response in humans (Thanavala et al, are amenable to transformation and metabolic engineering, for the 2005; McCormick et al., 2008) and mice (Daniell et al., 2001b; expression of humanize N-glycans (Cox et al., 2006). Besides, there Tregoning et al., 2005). However, about 100-fold of the vaccine has been recent revisiting of the rhizosecretion technology, a delivered by injection is required for oral administion (Streatfield and hydroponic production system, as a suitable, cost-effective production Howard, 2003). Notably, Franconi et al. (2006) and Massa et al. (2007) system, which also combines the advantage of confined environments reported that immunization with plant-produced antigen protected with the flexibility of adding the hydroponic medium directly to a the injected mice from tumour challenge with an E7-expressing chromatography column for affinity purification, thus allowing simple tumour cell line, while Koya et al. (2005) reported that mice injected and rapid production of high purity recombinant protein products and immunized with chloroplast-derived anthrax protective antigen

(Drake et al., 2009). Three affinity tags commonly used include His6, survived anthrax lethal toxin challenge. Also the Gal/GalNAc lectin of StrepII and Tandem Affinity Purification (TAP) tag (Terpe, 2003). An Entamoeba histolytica was found to be highly immunogenic in injected elastin-like polypeptide tag has also been optimized for expression mice (Chebolu and Daniell, 2007). There are several plant-produced and purification of recombinant proteins in plants (Conley et al., vaccine candidates, which are at different stages of clinical trials, as 2009). summarized in Table 1. It should be noted that the first veterinary vaccine, the NDV vaccine for poultry developed by Dow AgroSciences, 4. Overview of plant-derived recombinant proteins has been approved by the US Department of Agriculture (USDA) (http://www.dowagro.com/animalhealth). As such, the plant-based 4.1. Plant-derived vaccine antigens production processes are able to compete with conventional methods, breaking the limits of current standard production technologies and A vaccine is an antigenic preparation that improves immunity reaching new frontiers for the plant-based production of pharmaceu- against a particular disease. The development of non-replicating viral tical-grade proteins (Buonaguro and Butler-Ransohoff, 2010). protein subunit was inspired by the intrinsic risks involved in producing large amounts of vaccines through the traditional method 4.2. Plant-derived antibodies of using live attenuated or inactivated viruses (Varsani et al., 2003). Hence, there was a shift to the current production of subunit vaccines Antibodies or immunoglobulins (IgGs) are serum proteins that in bacteria, yeast, and mammalian cells. However, these conventional bind to target molecules with high specificity and are widely used for expression systems have some peculiar problems. As such, the idea of prevention, detection and treatment of diseases. Up till recently, using plants as a platform for production of vaccines was borne out of monoclonal antibodies were only being used to treat diseases such as its numerous potential advantages over the existing ones, including arthritis, cancer, immune and inflammatory diseases. Recombinant 216 O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222

Table 1 Plant-derived pharmaceuticals in clinical stages of development or on market. Information from Basaran and Rodríguez-Cerezo (2008), Kaiser (2008), Key et al. (2008), Spök et al. (2008), Sharma and Sharma (2009), Lau and Sun (2009), Obembe (2010), Faye and Gomord (2010). Updated from company websites.

Product Disease Plant Clinical trial status Company Source: URL/academic

Vaccines Hepatitis B antigen Hepatitis B Lettuce Phase I Thomas Jefferson University, USA Streatfield, 2006 (HBsAg) Potato Phase II Arizona State University Fusion proteins, including Rabies Spinach Phase I completed Thomas Jefferson University, USA www.labome.org/expert/usa/.../ epitopes from rabies hilary-koprowski-233492.html Cancer vaccine Non-Hodgkin's Tobacco Phase II Large Scale Biologya, USA http://www.gmo-safety.eu/article/483. lymphoma pharma-plants-status-report.html Vibrio cholerae Cholera Potato Phase I Arizona State University Tacket, 2005 Heat-labile toxin B subunit Diarrhea Maize Phase I ProdiGeneb, USA Tacket, 2005 of Escherichia coli Potato Phase I Arizona State University Capsid protein Norwalk Diarrhea Potato, Phase I Arizona State University Khalsa et al., 2004 virus Tomato Antigen Feline parvovirus Tobacco Advanced Large Scale Biologya, USA http://www.lsbc.com (Dogs) Antigen Papilloma virus Tobacco Early Large Scale Biologya, USA http://www.lsbc.com (Rabbit) HN protein of Newcastle Newcastle disease Tobacco suspension USDA Approved Dow Agro Sciences, http://www.dowagro.com/ disease virus (Poultry) cells USA animalhealth Viral vaccine mixture Diseases of horses, Tobacco suspension Phase I Dow Agro Sciences, http://www.dowagro.com/ dogs, and birds cells USA animalhealth Poultry vaccine Coccidiosis infection Canola Phase II Guardian Biosciences, Canada Basaran and Rodríguez-Cerezo (2008) Gastroenteritis virus Piglet gastroenteritis Maize Phase I ProdiGeneb, USA Basaran and Rodríguez-Cerezo (2008) (TGEV) capsid protein H5N1 vaccine candidate H5N1 pandemic Tobacco Phase I Medicago, USA http://www.medicago.com influenza

Antibodies CaroRX Dental caries Tobacco EU approved as Planet Biotechnology, USA http://www.planetbiotechnology.com/ medical advice DoxoRX Side-effects of Tobacco Phase I completed Planet Biotechnology, USA http://www.planetbiotechnology.com/ cancer therapy RhinoRX Common cold Tobacco Phase I completed Planet Biotechnology, USA http://www.planetbiotechnology.com/ Fv antibodies Non-Hodgkin's Tobacco Phase I Large Scale Biology, USA http://www.lsbc.coma lymphoma IgG (ICAM1) Common cold Tobacco Phase I Planet Biotechnology, USA http://www.planetbiotechnology.com/ Antibody against hepatitis B Vaccine purification Tobacco On market CIGB, Cuba Kaiser, 2008

Therapeutic human proteins Gastric lipase, Merispase® Cystic fibrosis Maize On market Meristem Therapeutics France http://www.meristem-therapeutics. com α-Galactosidase Fabry disease Tobacco Phase I Planet Biotechnology, USA http://www.planetbiotechnology.com/ Lactoferon™ Hepatitis B and C Duckweed Phase II Biolex, USA http://www.biolex.com/ (α-interferon) Fibrinolytic drug Blood clot Duckweed Phase I Biolex, USA http://www.biolex.com/ (thrombolytic drug) Human glucocerebrosidase Gaucher ‘s disease Carrot suspension Awaiting USDA's Protalix Biotherapeutics, http://www.protalix.com/ (prGCD) cells approval Israel glucocerebrosidase.html Insulin Diabetes Safflower Phase III SemBioSys, Canada http://www.sembiosys.com/ Apolipoprotein Cardiovascular Safflower Phase I SemBioSys, Canada http://www.sembiosys.com/

Nutraceuticals ISOkine™ , DERMOkine™ Human growth factor Barley On market ORF Genetics http://www.orfgenetics.com/ Human intrinsic factor, Vitamin B12 deficiency Arabidopsis On market Cobento Biotech AS http://www.cobento.dk/ Coban default.asp?id=76 Human lactoferrin Anti-infection, Rice Advanced, on market Ventria, USA http://www.ventriabio.com/ anti-inflammatory as fine chemical Human lysozyme Anti-infection, Rice Advanced, on market Ventria, USA http://www.ventria.com/ anti-inflammatory as fine chemical Immunosphere™ Food additive for Safflower Marketing expected SemBioSys, Canada http://www.sembiosys.com/ shrimps for 2010

a LSBC filed bankruptcy in 2006. b Prodigene has winded up activity.

antibodies have now been found to provide passive immunization alternatives for large scale production (Stoger et al., 2005b), as bulk against pathogens, as such are potentiated as promising alternatives production in the established production system- the mammalian to combat infectious diseases, especially in the face of increasing cells, presently poses difficult challenge, with respect to the microbial resistance to antibiotics and the emergence of new complexity of the product and the different processes involved, such pathogens (Casadevall, 1998). Though, there is increasing market as post-translational modifications and proteolysis. Plants do not only demand, however, the prevailing high cost of production is prevent- provide cheaper production platforms, as plant-derived antibodies ing their successful introduction into the health market, as therapy of would cost just 0.1–1% of the production cost of mammalian culture infectious diseases. These opposing factors, thus, call for cost-effective and 2–10% of microbial systems (Chen et al., 2005), but also can also O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222 217 assemble complex multimeric antibodies (Conrad and Fiedler, 1994), 4.4. Non-pharmaceutical plant-derived proteins as such can be engineered for the production of diverse tailor-made pharmaceutical proteins. Since the first recombinant antibodies were The non-pharmaceutical plant-derived proteins or industrial expressed in plants in 1989 (Hiatt et al., 1989), different moieties proteins, most of which are enzymes, including avidin, trypsin, ranging from single chain Fv fragments (ScFvs, which contain the aprotinin, β-glucuronidase, peroxidase, laccase, cellulase and others variable regions of the heavy and light chains joined by a flexible listed in Basaran and Rodríguez-Cerezo (2008) are now on the peptide linker) to Fab fragments (assembled light chains and market. The molecular farming of cell-wall deconstructing enzymes shortened heavy chains), small immune proteins (SIP), IgGs, and such as cellulases, hemicellulases, xylanases and ligninases, in chimeric secretory IgA and single-domain antibodies have been particular, holds great promise for the biofuel industry, with respect expressed as well (Ismaili et al., 2007; Xu et al., 2007). to the production of cellulosic ethanol (Sticklen, 2008; Mei et al., There are two main approaches that are being employed to 2009; Chatterjee et al., 2010), which was estimated to have the produce biologically active whole antibodies in plants. The first is potential of reducing greenhouse gas emissions by 100% compared to cross-pollination of individually transformed plants expressing light gasoline (Fulton et al., 2004). Other non-hydrolytic proteins, such as or heavy chains, resulting in high yield which reaches 1 to 5% of total the cell wall disrupting carbohydrate binding modules of cell wall plant protein (Hiatt et al., 1989; Ma et al., 1994). The second approach deconstructing enzymes, and the cell wall loosening proteins, the involves co-transformation of the heavy and light chain genes on a expansins, which have been implicated in enhancing the efficiency of single- (During et al., 1990), two- (Villani et al., 2008) or more wall degradation by disrupting the different polysaccharide networks, expression cassettes (Nicholson et al., 2005). One of such plant- thereby allowing increase accessibility of the hydrolytic enzymes to derived antibodies, a secretory antibody against a surface antigen of the substrate, have been previously demonstrated to alter cell wall Streptococcus mutans was actually found to be as effective as the structure (Obembe et al., 2007a; Obembe et al., 2007b), and as such original mouse IgG, in protecting against S. mutans colonization on have been recommended as candidate PMF proteins for the biofuel teeth, (Ma et al., 1998). The antibody has since been developed further industry (Obembe, 2010). by Planet Biotechnology, Inc. into a clinical product, CaroRX™, which Other potential non-pharmaceutical plant-derived technical pro- has recently received EU approval to be used as medical advice for the teins that are being explored and optimized for production include prevention of oral bacterial infection that contributes to dental caries biodegradable plastic-like compounds such as polyhydroxyalkanoate (Kaiser, 2008). Recently, a HIV-specific monoclonal antibody, which (PHA) copolymers, Poly (3-hydroxybutyrate) (PHB) and cyanophycin was produced in maize seeds, was found to be as active as its Chinese (Conrad, 2005; Matsumoto et al., 2009). It should be noted that thus hamster ovary-derived counterpart, in terms of antigen-binding far, only few plant-derived pharmaceuticals have been approved, and activity (Ramessar et al., 2008a, 2008b). There are five different fewer still are commercially available, majorly because of biosafety plant-derived monoclonal antibodies presently being tested in the concerns and stringent governmental regulations with respect to field clinical trials, as listed in Table 1. The sixth one, Avicidin was trials, good manufacturing practice (GMP) standards and pre-clinical withdrawn from the trials because of several side effects suffered by toxicity testing. the participants, even though it showed promising results against advanced colon and prostate cancer (Ma et al., 2005). The first plant- 5. Biosafety and regulatory issues made scFv monoclonal antibody, used in the production of a recombinant hepatitis B virus vaccine, has been commercialized in The general public concern about the potential health and Cuba (Pujol et al., 2005). environmental risks associated with the PMF crops (not the products) is being viewed at two levels; in that, not only are they engineered to 4.3. Therapeutic and nutraceutical proteins accumulate all sorts of proteins with medicinal properties at very high concentration, which may affect the host plants (Badri et al., 2009), The first therapeutic human protein to be expressed in plants was but also that their biologically active products are meant to elicit a human growth hormone (Barta et al., 1986). In 1990, human serum physiological responses in humans and in animals (Spök, 2007; Spök albumin, which is normally isolated from blood, was produced in et al., 2008). Additionally, there are specific concerns, including the transgenic tobacco and potato for the first time (Sijmons et al., 1990). risks of (i) co-mingling with food/feed crops and the resultant Since then, several human proteins have been expressed in the plants. economic risks to farmers and food industry, (ii) transgenes spread by These include epidermal growth factor (Wirth et al., 2004; Bai et al., pollen, seed or fruit dispersal, (iii) unintended exposures to non- 2007), α-, β- and γ-interferons, which are used in treating hepatitis B target organisms, particularly birds, insects and soil microorganisms, and C (Sadhu, and Reddy, 2003; Zhu et al., 2004; Arlen et al., 2007), and (iv) horizontal gene transfer by asexual means (Commandeur erythropoietin, which promote red blood cell production (Weise et al., et al., 2003; Andow et al., 2004; Wisner, 2005). 2007; Musa et al., 2009), interleukin, which is used in treating Crohn's Like for the GM food and feed crops, several regulations are being disease (Elias-Lopez et al., 2008; Fujiwara et al., 2010), insulin, which developed, to increase the biosafety of the plant bioreactors, even is used for treating diabetes (Nykiforuk et al., 2006), human though, one knows that, there is no fool-proof system, as there might glucocerebrosidase, which is used for the treatment of Gaucher's be some elements of human errors and natural accidents, which are disease in genetically engineered carrotcells (Shaaltiel et al., 2007) beyond control. and several others. Also contained in Table 1 is the list of some of these therapeutics, which are at various stages of clinical trials or at the 5.1. Problem of co-mingling and contamination of the food/feed chain verge of being commercialized. Antimicrobial nutraceutics, such as human lactoferrin and lyso- The problem of co-mingling and contamination of the food/feed zymes, have now been successfully produced in several crops (Huang chain is being prevented by the use of non-food and non-feed crops, et al., 2008; Stefanova et al., 2008), and now commercially available, such as tobacco, lemna, Arabidopsis, microalgae or mosses (Cox et al., only as fine chemicals (Table 1). Cobento Biotechnology, Denmark has 2006; Decker and Reski, 2008; Franklin and Mayfield, 2004; Tremblay just recently received approval for its Arabidopsis-derived human et al., 2010). However, when it is desirable to use food or feed crops, as intrinsic factor (Key et al., 2008), which is to be used against vitamin in the case of monoclonal antibodies and oral vaccines, strict physical B12 deficiency and now commercially available as Coban (Sharma and agronomic confinement and containment strategies are being applied Sharma, 2009). Other nutraceutical products at various stages of to avoid co-mingling. These include growing in small restricted and development are listed in Table 1. isolated areas, remote from the agricultural crops to avoid mechanical 218 O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222 mixing (Ma et al., 2005; US Department of Agriculture, 2006; Spök the regulatory guidelines on GM crops in general as being relaxed or et al., 2008), crop destruction and post-planting field cleaning, compromised, which explains why some unapproved GM foods still planting at different periods to ensure harvesting at different periods manage to find their ways to the dining tables in the US and most from other crops intended for use as food and feed (Spök, 2007), the unexpectedly in Europe. This calls for stricter compliance with good use of non-commercial varieties or plant containing visible marks manufacturing practice standards, all through the stages of develop- (Biemelt and Sonnewald, 2005), prohibition of trade in seeds and ment, in order to ensure the delivery of clinical grade plant-derived viable plants (Spök et al., 2008), and the use of dedicated machineries products. and facilities (US Department of Agriculture, 2006). As it is very fi dif cult to avoid admixtures of GM and non-GM crops, even with 6. Concluding remarks confinement (USDA/APHIS, 2006b), threshold limits of accidental contaminations are now being introduced, for example in Europe, No doubt the PMF industry is here to stay, considering the quantum 0.5% presence of GM in non-GM or feed is allowed (European of advances that have been made in the past decade alone. Though, there Parliament, 2003) and for non hazardous, non-pharmaceutical plant- had been some disappointing incidence at some points, which led to the made products, the threshold limit of contamination is set at 0.9% lull in the industry, and even to the folding up of some of the big players, (Spök, 2007). including Prodigene, we do share the same sentiment with Kaiser (2008) that exciting times are now returning to the industry, as a 5.2. The problem of gene spread and unintended exposures number of new players are now picking up interest and entering into the field. One such player is the multinational company Bayer, which is The biosafety strategies being used to address the problem of gene collaborating with a number of biotech companies and has recently spread and unintended exposures of the plant bioreactors, irrespective of bought the German company, Icon Genetics that developed the the status (food or non-food crop), include the use of closed isolated MagnICON technology. Also worth mentioning, is the research program containment facilities, such as greenhouses (Menassa et al., 2001), of the EU-funded Pharma-Planta consortium, which is devoted to glasshouses, hydroponics (Commandeur et al., 2003; Drake et al., 2009) developing and ultimately producing recombinant pharmaceuticals in and plant cell suspension cultures (www.protalix.com)(Franconi et al., plants, for humanitarian purpose! With their recent successful 2010; Plasson et al., 2009), the use of biological containment, such as self- development of a cost-effective seed-based production technology for fl pollinating species (cleistogamous lines) (Le on et al., 2009), chloroplast a vaginal protection microbicide to prevent heterosexual HIV transmis- transformation (Svab and Maliga, 2007), the use of male-sterile sion, the stage in now set for the deployment of the same technology to transgenic plants (Gils et al., 2008), the use of sexually incompatible produce other therapeutic bio-molecules on a large scale, which could crop with wild relatives, the production of non-germinating seeds or be made more widely available and cheaper than presently possible non-sprouting tubers/bulbs (through the use of a technology known as (Ramessar et al., 2008a, 2008b). In 2009, another International Research “ ” Genetic use restriction technologies (GURTs )(Hills et al., 2007). Other Group (consisting of Universities, Research Institutes and new biotech strategies being pursued to minimize the gene spread and exposure to companies) successfully accumulated over 1 g/kg of a red algal protein, other organisms include cytoplasmic male sterility (Chase, 2006), a lectin known as griffithsin (GRFT-P) in Nicotiana benthamiana leaves engineered parthenocarpy and apomixis (Rotino et al., 2005; Sandhu (O'Keefe et al., 2009). This protein, in addition to exhibiting broad- et al., 2009), transgene excision (Gidoni et al., 2008), engineering the spectrum activity against a wide range of HIV strains and similar viruses fi plants for organ- or tissue-speci cexpressionofthetransgene(for causing sexually transmitted infections, represents the most potent HIV example in the fruits) (He et al., 2008), and the use of inducible promoters entry inhibitor to date (Zeitlin et al., 2009; O'Keefe et al., 2009). Having that respond to external stimuli or chemicals can be exploited to regulate met all the requirements to be used as a microbicide, GRFT-P is now gene expression at some point after harvest (Corrado and Karali, 2009), being validated as a candidate protein microbicide for clinical trials especially as it has been shown that certain plant-derived insecticides and (O'Keefe et al., 2009) and it is hoped that the recent results on efficacy biopharmaceutical proteins can remain stable in the soil for a long period dilution of candidate microbicide (Mâsse et al., 2009) would be useful (Basaran and Rodríguez-Cerezo, 2008). Another strategy being deployed for successful trials. It is envisioned that all of these developments in for curtailing the effect of gene spread is transgenic mitigation, which PMF would begin to attract newer stakeholders to join in, especially for fl does not by itself prevent transgene ow from transgenic crops to clinical development of large volume biologics, which are presently very ‘ ’ nontransgenic crops or wild relatives, but mitigates the effects of such expensive to manufacture with the conventional manufacturing fl gene ow, i.e. its goal is to prevent the establishment of the transgene in practice. Most of all, the overall prospects of plant-based molecular volunteer populations or in populations of wild relatives if hybridization farming industry will depend on improved public perception of the can occur (Gressel and Valverde, 2009). technology and the products, especially as the industry improves its level of compliance with the regulations, and as there are many 5.3. Risk of horizontal gene transfer successful clinical trials and approvals of the first set of these plant- derived human pharmaceuticals. It is also envisioned that the public The risk of horizontal gene transfer from plants to microbes, perception would be all the more positive with the recent European especially in the case of using antibiotic resistance genes, is generally Union's preparation for “pharmed drugs” (Gilbert, 2009; Rybicki, 2010). believed to be extremely low, as there has been no report of such incidence to date. More so, it is widely known that plants, including Acknowledgement the edible ones, naturally harbor loads of bacteria with antibiotic- resistant genes (Nielsen et al., 1998). Olawole O. Obembe would like to thank the International Centre It should be noted that most of the aforementioned biosafety for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy for strategies have been motivated by regulatory policies and guidelines of sponsoring his post-doctoral training on Chloroplast Engineering. the different countries involved in the research, development and industrial applications of the PMF technologies. However, it has been admitted that the enforcement of the regulations is still leaky, as it is References difficult to monitor all the different stages of development and processing of the plant-derived products, as such there exists some Ananiev EV, Wu C, Chamberlin MA, Svitashev S, Schwartz C, Gordon-Kamm W, et al. Artificial chromosome formation in maize (Zea mays L.). Chromosoma 2009;118:157–77. laxities in the level of compliance on the part of the operators, which go Andow D, Daniell H, Gepts P, Lamkey K, Nafziger E, Strayer D. A growing concern. undetected by the regulatory agencies. The civil society now perceives Protecting the food supply in an era of pharmaceutical and industrial crops. . Dec. O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222 219

Available from:In: Andow D, editor. Technical ReportUnion of Concerned De Jaeger G, Scheffer S, Jacobs A, Zambre M, Zobell O, Goossens A, et al. Boosting Scientists; 2004. http://www.biosafety-info.net/file_dir/3079248859c6963300. heterologous protein production in transgenic dicotyledonous seeds using pdf. accessed 28/8/09. Phaseolus vulgaris regulatory sequences. Nat Biotechnol 2002;20:1265–8. Arlen PA, Falconer R, Cherukumilli S, Cole A, Cole AM, Oishi KK, et al. Field production De Muynck B, Navarre C, Nizet Y, Stadlmann J, Boutry M. Different subcellular and functional evaluation of chloroplast derived interferon-α2b. Plant Biotechnol J localization and glycosylation for a functional antibody expressed in Nicotiana 2007;5:511–25. tabacum plants and suspension cells. Transgenic Res 2009;18:467–82. Aviezer D, Brill-Almon E, Shaaltiel Y, Hashmueli S, Bartfeld D, Mizrachi S, et al. A plant- De Muynck B, Navarre C, Boutry M. Production of antibodies in plants: status after derived recombinant human glucocerebrosidase enzyme—a preclinical and phase I twenty years. Plant Biotechnol J 2010;8:529–63. investigation. PLoS ONE 2009;4:e4792. De Paepe A, De Buck S, Hoorelbeke K, Nolf J, Peck I, Depicker. A high frequency of single Badri MA, Rivard D, Coenen K, Michaud D. Unintended molecular interactions in copy T-DNA transformants by floral dip in CRE-expressing Arabidopsis plants. Plant transgenic plants expressing clinically useful proteins: the case of bovine aprotinin J 2009;59:517–27. traveling the potato leaf cell secretory pathway. Proteomics 2009;9:746–56. Decker EL, Reski R. The moss bioreactor. Curr Opin Plant Biol 2004;7:166–70. Bai JY, Zeng L, Hu YL, Li YF, Lin ZP, Shang SC, et al. Expression and characteristic of Decker EL, Reski R. Current achievements in the production of complex biopharma- synthetic human epidermal growth factor (hEGF) in transgenic tobacco plants. ceuticals with moss bioreactors. Bioprocess Biosyst Eng 2008;31:3–9. Biotechnol Lett 2007;29:2007–12. Drake PM, Chargelegue DM, Vine ND, van Dolleweerd CJ, Obregon P, Ma JKC. Barta A, Sommengruber K, Thompson D, Hartmuth K, Matzke MA, Matzke AJM. The Rhizosecretion of a monoclonal antibody protein complex from transgenic tobacco expression of a napoline synthase human growth hormone chimeric gene in roots. Plant Mol Biol 2003;52:233–41. transformed tobacco and sunflower callus tissue. Plant Mol Biol 1986;6:347–57. Drake PM, Barbi T, Sexton A, McGowan E, Stadlmann J, Navarre C, et al. Development of Basaran P, Rodríguez-Cerezo E. Plant molecular farming: opportunities and challenges. rhizosecretion as a production system for recombinant proteins from hydroponic Crit Rev Biotechnol 2008;28:153–72. cultivated tobacco. FASEB J 2009;23:3581–9. Benchabane M, Goulet C, Rivard D, Faye L, Gomord V, Michaud D. Preventing unintended During K, Hippe S, Kreutzaler F, Schell J. Synthesis and self-assembly of a functional proteolysis in plant protein biofactories. Plant Biotechnol J 2008;6:633–48. monoclonal antibody in transgenic Nicotiana tobacum. Plant Mol Biol 1990;15: Bevan MW, Flavell RB, Chilton MD. A chimaeric antibiotic resistance gene as a selectable 281–93. marker for plant cell transformation. Nature 1983;304:184–7. Elias-Lopez AL, Marquina B, Gutierrez-Ortega A, Aguilar D, Gomez-Lim M, Hernández- Biemelt S, Sonnewald U. Molecular farming in plants. Nature encyclopedia of life Pando R. Clinical and experimental immunology transgenic tomato expressing sciences. London: Nature Publishing Group; 2005 http://www.els.net/, doi: interleukin-12 has a therapeutic effect in a murine model of progressive pulmonary 10.1038/npg.els.0003365. tuberculosis. Clin Exp Immunol 2008;154:123–33. Biemelt S, Sonnewald U, Galmbacher P, Willmitzer L, Müller M. Production of human European Parliament. Regulation (EC) No 1829/2003 of the European Parliament and of papillomavirus type 16 virus-like particles in transgenic plants. J Virol 2003;77: the council of 22 September 2003 on genetically modified food and feed. Available 9211–20. from:Off J Eur Union 2003;268:1-23. http://eurlex.europa.eu/LexUriServ/site/en/ Bock R. Plastid biotechnology: prospects for herbicide and insect resistance, metabolic oj/2003/l_268/l_26820031018en00010023.pdf accessed 20/8/09. engineering, and molecular farming. Curr Opin Biotechnol 2007;18:100–6. Faye L, Gomord V. Success stories in molecular farming—a brief overview. Plant Boothe J, Nykiforuk C, Shen Y, Zaplachinski S, Szarka S, Kuhlman P, et al. Seed-based Biotechnol J 2010;8:525–8. expression systems for plant molecular farming. Plant Biotechnol J 2010;8:588–606. Fischer R, Emans NJ, Twyman RM, Schillberg S. Molecular farming in plants: technology Bosch D, Schots A. Plant glycans: friend or foe in vaccine development? Expert Rev platforms. In: Goodman RB, editor. Encyclopedia of plant and crop science. New Vaccin 2010;9:835–42. York: Marcel Dekker; 2004. p. 753–6. Breyer D, Goossens M, Herman P, Sneyers M. Biosafety considerations associated with Floss DM, Sack M, Arcalis E, Stadlmann J, Quendler H, Rademacher T, et al. Influence molecular farming in genetically modified plants. J Med Plants Res 2009;3:825–38. of elastin-like peptide fusions on the quantity and quality of a tobacco-derived Buonaguro FM, Butler-Ransohoff J-E. PharmaPlant: the new frontier in vaccines. Expert human immunodeficiency virus-neutralizing antibody. Plant Biotechnol J 2009;7: Rev Vaccin 2010;9:805–7. 899–913. Cardi T, Lenzi P, Maliga P. Chloroplasts as expression platform for plant-produced Franconi R, Massa S, Illiano E, Mullar A, Cirilli A, Accardi L, et al. Exploiting the plant vaccines. Expert Rev Vaccin 2010;9:893–911. secretory pathway to improve the anticancer activity of a plant-derived HPV16 E7 Casadevall A. Antibody-based therapies as anti-infective agents. Expert Opin Emerg vaccine. Int J Immunopathol Pharmacol 2006;19:187–97. Drugs 1998;7:307–21. Franconi R, Demurtas OC, Massa S. Plant-derived vaccines and other therapeutics Castilho A, Strasser R, Stadlmann J, Grass J, Jez J, Gattinger P. In planta protein sialylation produced in contained systems. Expert Rev Vaccin 2010;9:877–92. through overexpression of the respective mammalian pathway. J Biol Chem Franklin SE, Mayfield SP. Prospects for molecular farming in the green alga 2010;285:15923–30. Chlamydomonas. Curr Opin Plant Biol 2004;7:159–65. Chase CD. Genetically engineered cytoplasmic male sterility. Trends Plant Sci 2006;11:7–9. Fujiwara Y, Aiki Y, Yang L, Takaiwa F, Kosaka, Tsuji NM, et al. Extraction and purification Chatterjee A, Das NC, Raha S, Babbit R, Huang Q, Zeitlin D, et al. Production of xylanase of human interleukin-10 from transgenic rice seeds. Protein Expr Purif 2010;72: in transgenic tobacco for industrial use in bioenergy and biofuel applications. In 125–30. Vitro Cell Dev Biol Plant 2010;46:198–209. Fulton L, Howes T, Hardy J. Biofuels for transport—an international perspective. Chebolu S, Daniell H. Stable expression of Gal/GalNAc lectin of Entamoeba histolytica in Available from:Paris, France: International Energy Agency; 2004. http://www.iea. transgenic chloroplasts and immunogenicity in mice towards vaccine development org/textbase/nppdf/free/2004/biofuels2004.pdf. accessed 18/9/09. for amoebiasis. Plant Biotechnol J 2007;5:230–9. Gidoni D, Srivastava V, Carmi N. Site-specific excisional recombination strategies for Chen M, Liu M, Wang Z, Song J, Qi Q, Wang PG. Modification of plant N-glycans elimination of undesirable transgenes from crop plants. InVitro Cell Dev Biol Plant processing: the future of producing therapeutic proteins in transgenic plants. Med 2008;44:457–67. Res Rev 2005;25:343–60. Gilbert N. Europe prepares for drugs from GM plants. Nature 2009;460:791. Chikwamba R, McMurray J, Shou H, Frame B, Pegg SE, Scott P, et al. Expression of a Gils M, Marillonnet S, Werner S, Grätzner R, Giritch A, Engler C, et al. A novel hybrid synthetic E. coli heat-labile enterotoxin B sub-unit (LT-B) in maize. Mol Breed seed system for plants. Plant Biotechnol J 2008;6:226–35. 2002;10:253–65. Giorgi C, Franconi R, Rybicki EP. Human papillomavirus vaccines in plant. Expert Rev Commandeur U, Twyman RM, Fischer R. The biosafety of molecular farming in plants. Vaccin 2010;9:913–24. AgBiotechNet 2003;5:ABN110. Giritch A, Marillonnet S, Engler C, van Eldik G, Botterman J, Klimyuk V, et al. Rapid high- Conley AJ, Joensuu JJ, Jevnikar AM, Menassa R, Brandle JE. Optimization of elastin-like yield expression of full-size IgG antibodies in plants coinfected with noncompeting polypeptide fusions for expression and purification of recombinant proteins in viral vectors. Proc Natl Acad Sci USA 2006;103:14701–6. plants. Biotechnol Bioeng 2009;103:562–73. Gleba Y, Klimyuk V, Marillonnet S. Magnifection—a new platform for expressing Conrad U. Polymers from plants to develop biodegradable plastics. Trends Plant Sci recombinant vaccines in plants. Vaccine 2005;23:2042–8. 2005;10:511–2. Gomord V, Fitchette A-C, Menu-Bouaouiche L, Saint-Jore-Dupas C, Plasson C, Michaud Conrad U, Fiedler U. Expression of engineered antibodies in plant cells. Plant Mol Biol D, et al. Plant-specific glycosylation patterns in the context of therapeutic protein 1994;26:1023–30. production. Plant Biotechnol J 2010;8:564–87. Conrad U, Fiedler U. Compartment-specific accumulation of recombinant immunoglo- Goulet C, Benchabane M, Anguenot R, Brunelle F, Khalf M, Michaud D. A companion bulins in plant cells: an essential tool for antibody production and immunomo- protease inhibitor for the protection of cytosol-targeted recombinant proteins in dulation of physiological functions and pathogen activity. Plant Mol Biol 1998;38: plants. Plant Biotechnol J 2010;82:142–54. 101–9. Gressel J, Valverde BE. A strategy to provide long-term control of weedy rice while Corrado G, Karali M. Inducible gene expression systems and plant biotechnology. mitigating herbicide resistance transgene flow, and its potential use for other crops Biotechnol Adv 2009;27:733–43. with related weeds. Pest Manag Sci 2009;65:723–31. Cox KM, Sterling JD, Regan JT, Gasdaska JR, Frantz KK, Peele CG, et al. Glycan Harvey AJ, Speksnijder G, Baugh LR, Morris JA, Ivarie R. Expression of exogenous protein optimization of a human monoclonal antibody in the aquatic plant Lemna minor. in the egg white of transgenic chickens. Nat Biotechnol 2002;20:396–9. Nat Biotechnol 2006;24:1591–7. He Z, Jiang XL, Qi Y, Di QL. Assessment of the utility of the tomato fruit-specificE8 D'Aoust M-A, Couture MM-J, Charland N, Tre´panier S, Landry N, Ors F, et al. The promoter for driving vaccine antigen expression. Genetica 2008;133:207–14. production of hemagglutinin-based virus-like particles in plants: a rapid, efficient Hiatt A, Cafferkey R, Bowdish K. Production of antibodies in transgenic plants. Nature and safe response to pandemic influenza. Plant Biotechnol J 2010;8:607–9. 1989;342:76–8. Daniell H, Lee SB, Panchal T, Wiebe PO. Expression of the native cholera toxin B subunit Hills MJ, Hall L, Arnison PG, Good AG. Genetic use restriction technologies (GURTs): gene and assembly as functional oligomers in transgenic tobacco chloroplasts. J Mol strategies to impede transgene movement. Trends Plant Sci 2007;12:177–83. Biol 2001a;311:1001–9. Hohe A, Decker E, Gorr G, Schween G, Reski R. Tight control of growth and cell Daniell H, Streatfield SJ, Wycoff K. Medical molecular farming: production of antibodies, differentiation in photoautotrophically growing moss (Physcomitrella patens) biopharmaceuticals, and edible vaccines in plants. Trends Plant Sci 2001b;6:219–26. bioreactor cultures. Plant Cell Rep 2002;20:1135–40. 220 O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222

Hood EE, Witcher DR, Maddock S, Meyer T, Baszczynski C, Bailey M, et al. Commercial Ma JKC, Lehner T, Stabila P, Fux CI, Hiatt A. Assembly of monoclonal antibodies with production of avidin from transgenic maize: characterization of transformant, IgG1 and IgA heavy chain domains in transgenic tobacco plants. Eur J Immunol production, processing, extraction and purification. Mol Breed 1997;3:291–306. 1994;24:131–8. Horn ME, Woodard SL, Howard JA. Plant molecular farming: systems and products. Ma JKC, Hikmat BY, Wycoff K, Vine ND, Chargelegue D, Yu L, et al. Characterization of a Plant Cell Rep 2004;22:711–20. recombinant plant monoclonal secretory antibody and preventive immunother- Huang N, Bethell D, Card C, Cornish J, Marchbank T, Wyatt D, et al. Bioactive apy. Nat Med 1998;4:601–6. recombinant human lactoferrin, derived from rice. In Vitro Cell Dev Biol 2008;44: Ma JKC, Barros E, Bock R, Christou P, Dale PJ, Dix PJ, et al. Molecular farming for new 464–71. drugs and vaccine. EMBO Rep 2005;6:593–9. Hull AK, Criscuolo CJ, Mett V, Groen H, Steeman W, Westra H, et al. Human-derived, Maclean J, Koekemoer M, Olivier AJ, Stewart D, Hitzeroth II, Rademacher T, et al. plant-produced monoclonal antibody for the treatment of anthrax. Vaccine Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: 2005;23:2082–6. comparison of the suitability of different HPV-16L1 gene variants and different cell- Ismaili A, Jalali JM, Rasaee MJ, Rahbarizadeh F, Forouzandeh-Moghadam M. Memari HR compartment localization. J Gen Virol 2007;88:1460–9. production and characterization of anti-(mucin MUC1) single-domain antibody in Maggio C, Barbante A, Ferro F, Frigerio L, Pedrazzini E. Intracellular sorting of the tobacco (Nicotiana tobacum cultivar Xanthi). Biotechnol Appl Biochem 2007;47:11–9. tailanchored protein cytochrome b5 in plants: a comparative study using different Jones DN, Kroos R, Anema B, van Montfort B, Vooys A, van der Kraats S. High-level isoforms from rabbit and Arabidopsis. J Exp Bot 2007;58:1365–79. expression of recombinant IgG in the human cell line per.c6. Biotechnol Prog Mainieri D, Rossi M, Archinti M, Bellucci M, De Marchis F, Vavassori S, et al. A new 2003;19:163–8. recombinant storage protein constructed using maize gamma-zein and bean Kaiser J. Is the drought over for pharming? Science 2008;320:473–5. phaseolin. Plant Physiol 2004;136:3447–56. Kang JS, Frank J, Kang CH, Kajiura H, Vikram M, Ueda A. Salt tolerance of Arabidopsis Marquet-Blouin E, Bouche FB, Steinmetz A, Muller CP. Neutralizing immunogenicity of thaliana requires maturation of N-glycosylated proteins in the Golgi apparatus. transgenic carrot (Daucus carota L.)-derived measles virus hemagglutinin. Plant Proc Natl Acad Sci USA 2008;105:5933–8. Mol Biol 2003;51:459–69. Kapila J, De Rycke R, van Montagu M, Angenon G. An agrobacterium-mediated transient Mason HS, Ball JM, Shi JJ, Jiang X, Estes MK, Arntzen CJ. Expression of Norwalk virus gene expression system for intact leaves. Plant Sci 1997;122:101–8. capsid protein in transgenic tobacco and potato and its oral immunogenicity in Kawaguchi R, Bailey-Serres J. Regulation of translational initiation in plants. Curr Opin mice. Proc Natl Acad Sci USA 1996;93:5335–40. Plant Biol 2002;5:460–5. Mason HS, Warzecha H, Mor T, Arntzen CJ. Edible plant vaccines: applications for Key S, Ma JKC, Drake PMW. Genetically modified plants and human health. J R Soc Med prophylactic and therapeutic molecular medicine. Trends Mol Med 2002;8: 2008;101:290–8. 324–9. Khalsa G, Mason HS, Arntzen CJ. Plant-derived vaccines: progress and constrains. In: Massa S, Franconi R, Brandi R, Muller A, Mett V, Yusibov V, et al. Anti-cancer activity of Fischer R, Schillberg S, editors. Molecular farming: plant-made pharmaceuticals plant-produced HPV16 E7 vaccine. Vaccine 2007;25:3018–21. and technical proteins. Weinheim, Germany: Wiley-VCH; 2004. p. 135–58. Mâsse BR, Boily M-C, Dimitrov D, Desai K. Efficacy dilution in randomized Kim TG, Baek M, Lee EK, Kwon T-H, Yang M-S. Expression of human growth hormone in placebo-controlled vaginal microbicide trials. Emerg Themes Epidemiol 2009;6, transgenic rice cell suspension culture. Plant Cell Rep 2008;27:885–91. doi:10.1186/1742-7622-6-5. Knäblein J. Plant-based expression of biopharmaceuticals. In: Meyers RA, editor. Masumura T, Morita S, Miki Y, Kurita A, Morita S, Shirono H, et al. Production of biologically Encyclopedia of molecular cell biology and molecular medicine. Weinheim: Wiley- active human interferon-α in transgenic rice. Plant Biotechnol 2006;23:91–7. VCH Verlag GmbH & Co. KGaA; 2005. p. 386–407. Matsumoto K, Murata T, Nagao R, Nomura CT, Arai S, Arai Y, et al. Production of Komarnytsky S, Borisjuk N, Yakoby N, Garvey A, Raskin I. Cosecretion of protease shortchain-length/medium-chain-length polyhydroxyalkanoate (PHA) copolymer inhibitor stabilizes antibodies produced by plant roots. Plant Physiol 2006;141: in the plastid of Arabidopsis thaliana using an engineered 3-ketoacyl-acyl carrier 1185–93. protein synthase III. Biomacromolecules 2009;10:686–90. Komarova TV, Baschieri S, Donini M, Marusic C, Benvenuto E, Dorokhov YL. Transient Mayfield SP, Franklin SE. Expression of human antibodies in eukaryotic microalgae. expression systems for plant-derived biopharmaceuticals. Expert Rev Vaccin Vaccine 2005;23:1828–32. 2010;9:859–76. McCormick AA, Reddy S, Reinl SJ, Cameron TI, Czerwinkski DK, Vojdani F, et al. Plant- Koprivova A, Stemmer C, Altmann F, Hoffmann A, Kopriva S, Gorr G, et al. Targeted produced idiotype vaccines for the treatment of non-Hodgkin's lymphoma: safety knockouts of Physcomitrella lacking plant-specific immunogenic N-glycans. Plant and immunogenicity in a phase I clinical study. Proc Natl Acad Sci USA 2008;105: Biotechnol J 2004;2:517–23. 10131–6. Koya V, Moayeri M, Leppla SH, Daniell H. Plant-based vaccine: mice immunized with Mei C, Park SH, Sabzikar R, Ransom C, Qi C, Sticklen M. Green tissue-specific production chloroplast-derived anthrax protective antigen survive anthrax lethal toxin of a microbial endo-cellulase in maize (Zea mays L.) endoplasmic-reticulum and challenge. Infect Immun 2005;73:8266–74. mitochondria converts cellulose to fermentable sugars. J Chem Technol Biotechnol Krapp S, Mimura Y, Jefferis R, Huber R, Sondermann P. Structural analysis of human IgG- 2009;84:689–95. Fc glycoforms reveals a correlation between glycosylation and structural integrity. J Menassa R, Nguyen V, Jevnikar A, Brandle JA. Self-contained system for the field Mol Biol 2003;325:979–89. production of plant recombinant interleukin-10. Mol Breed 2001;8:177–85. Kumar SGB, Ganapathi TR, Bapat VA. Edible vaccines: current status and future Mett V, Lyons J, Musiychuk, Chichester JA, Brasil T, Couch R, et al. A plant-produced prospects. Physiol Mov Biol Plants 2004;10:37–47. plague vaccine candidate confers protection to monkeys. Vaccine 2007;25:3014–7. Kumar GBS, Ganapathi TR, Revathi CJ, Srinivas L, Bapat VA. Expression of hepatitis B Meyers B, Zaltsman A, Lacroix B, Kozlovsky SV, Krichevsky A. Nuclear and plastid surface antigen in transgenic banana plants. Planta 2005;222:484–93. genetic engineering of plants: comparison of opportunities and challenges. Kusnadi AR, Hood EE, Witcher DR, Howard JA, Nikolov ZL. Production and purification Biotechnol Adv 2010, doi:10.1016/j.biotechadv.2010.05.022. of two recombinant proteins from transgenic corn. Biotechnol Prog 1998;14: Milne R. Public attitudes toward molecular farming in the UK. AgBioForum 2008;11: 149–55. 106–13. Lamphear BJ, Streatfield SJ, Jilka JM, Brooks CA, Barker DK, Turner DD, et al. Delivery of Mishra S, Yadav DK, Tuli R. Ubiquitin fusion enhances cholera toxin B subunit subunit vaccines in maize seed. J Control Release 2002;85:169–80. expression in transgenic plants and the plant-expressed protein binds GM1 Lau OS, Sun SSM. Plant seeds as bioreactors for recombinant protein production. receptors more efficiently. J Biotechnol 2006;127:95-108. Biotechnol Adv 2009;27:1015–22. Moloney MM, Siloto RM. Modified oleosins. US 10/561; 2004. p. 178. Lauterslager TGM, Florack DEA, Van der Wal TJ, Molthoff JW, Langeveld JP, Bosch D, Moravec T, Schmidt MA, Herman EM, Woodford-Thomas T. Production of Escherichia et al. Oral immunization of naïve and primed animals with transgenic potato tubes coli heat labile toxin (LT) B subunit in soybean seed and analysis of its expressing LT-B. Vaccine 2001;19:2749–55. immunogenicity as an oral vaccine. Vaccine 2007;25:1647–57. Leflon M, Hüsken A, Njontie C, Kightley S, Pendergrast D, Pierre J, et al. Stability of the Musa TA, Hung CY, Darlington DE, Sane DC, Xie J. Overexpression of human erythropoietin cleistogamous trait during the flowering period of oilseed rape. Plant Breed in tobacco does not affect plant fertility or morphology. Plant Biotechnol Rep 2009;3: 2009;129:13–8. 157–65. Lentz EM, Segretin ME, Morgenfeld MM, Wirth SA, Santos MJD, Mozgovoj MV, et al. Nagaya S, Kawamura K, Shinmyo A, Kato K. The HSP terminator of Arabidopsis thaliana High expression level of a foot and mouth disease virus epitope in tobacco increases gene expression in plant cells. Plant Cell Physiol 2010;51:328–32. transplastomic plants. Planta 2010;231:387–95. Nara Y, Kurata H, Seki M, Taira K. Glucocorticoid-induced expression of a foreign gene Leon-Banares R, Gonzalez-Ballester D, Galvan A, Fernandez E. Transgenic microalgae as by the GVG system in transformed tobacco BY-2 cells. Biochem Eng J 2000;6: green cell-factories. Trends Biotechnol 2004;22:45–52. 185–91. Liénard D, Sourrouille C, Gomord V, Faye L. Pharming and transgenic plants. Biotechnol Nicholson L, Gonzalez-Melendi P, van Dolleweerd C, Tuck H, Perrin Y, Ma JK, et al. A Ann Rev 2007;13:115–47. recombinant multimeric immunoglobulin expressed in rice shows assembly- Ling H-Y, Pelosi A, Walmsley AM. Current status of plant-made vaccines for veterinary dependent subcellular localization in endosperm cells. Plant Biotechnol J 2005;3: purposes. Expert Rev Vaccin 2010;9:971–82. 115–27. Loos A, Van Droogenbroeck B, Hillmer S, Grass J, Kunert R, Cao J, et al. Production of Nielsen KM, Bones A, Smalla K, van Elsas JD. Horizontal gene transfer from transgenic monoclonal antibodies with a controlled N-glycosylation pattern in seeds of plants to terrestrial bacteria—a rare event? FEMS Microbiol Rev 1998;22:79-103. Arabidopsis thaliana. Plant Biotechnol J 2010 (early view, article online in advance Nochi T, Takagi H, Yuki Y, Yang L, Masumura T, Mejima M, et al. Rice-based mucosal of print). doi:10.1111/j.1467-7652.2010.00540.x. vaccine as a global strategy for cold-chain- and needle-free vaccination. Proc Natl Lowe JA, Jones P. Biopharmaceuticals and the future of the pharmaceutical industry. Acad Sci USA 2007;104:10986–91. Curr Opin Drug Discov Dev 2007;10:513–4. Nykiforuk CL, Boothe JG, Murray EW, Keon RG, Goren HJ, Markley NA, et al. Transgenic Lu J, Sivamani E, Azhakanandam K, Samadder P, Li X, Qu R. Gene expression expression and recovery of biologically active recombinant human insulin from enhancement mediated by the 5′ UTR intron of the rice rubi3 gene varied Arabidopsis thaliana seeds. Plant Biotechnol J 2006;4:77–85. remarkably among tissues in transgenic rice plants. Mol Genet Genomics Nykiforuk CL, Shen Y, Murray EW, Boothe JG, Busseuil D, Rhéaume E, et al. Expression 2008;279:563–72. and recovery of biologically active recombinant apolipoprotein AI (Milano) from O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222 221

transgenic safflower (Carthamus tinctorius) seeds. Plant Biotechnol J 2010 (Early Schillberg S, Zimmermann S, Voss A, Fischer R. Apoplastic and cytosolic expression of view, article online in advance of print). doi:10.1111/j.1467-7652.2010.00546.x. full-size antibodies and antibody fragments in Nicotiana tabacum. Transgenic Res Obembe OO. The plant biotechnology flight: is Africa on board? Afri J Biotechnol 1999;8:255–63. 2010;9:4300–8. Schillberg S, Twyman RM, Fischer R. Opportunities for recombinant antigen and Obembe OO, Jacobsen E, Visser RGF, Vincken J-P. Expression of an expansin carbohydrate- antibody expression in transgenic plants—technology assessment. Vaccine binding module affects xylem and phloem formation. Afri J Biotechnol 2007a;6: 2005;23:1764–9. 1608–16. Shaaltiel Y, Bartfeld D, Hashmueli S, Baum G, Brill-Almon E, Galili G, et al. Production of Obembe OO, Jacobsen E, Timmers J, Gilbert H, Blake AW, Knox JP, et al. Promiscuous, non- glucocerebrosidase with terminal mannose glycans for enzyme replacement catalytic, tandem carbohydrate-binding modules modulate the cell-wall structure and therapy of Gaucher's disease using a plant cell system. Plant Biotechnol J 2007;5: development of transgenic tobacco (Nicotiana tabacum) plants. J Plant Res 2007b;120: 579–90. 605–17. Sharma AK, Sharma MK. Plants as bioreactors: recent developments and emerging Oey M, Lohse M, Kreikemeyer B, Bock R. Exhaustion of the chloroplast protein synthesis opportunities. Biotechnol Adv 2009;27:811–32. capacity by massive expression of a highly stable protein antibiotic. Plant J 2009;57: Sharma MK, Singh NK, Jani D, Sisodia R, Thungapathra M, Gautam JK, et al. Expression of 436–45. toxin co-regulated pilus subunit A (TCPA) of Vibrio cholerae and its immunogenic O'Keefe BR, Vojdani F, Buffa V, Shattock RJ, Montefiori DC, Bakke J, et al. Scaleable epitopes fused to choleratoxin B subunit in transgenic tomato (Solanum manufacture of HIV-1 entry inhibitor griffithsin and validation of its safety and lycopersicum). Plant Cell Rep 2008;27:307–18. efficacy as a topical microbicide component. Proc Natl Acad Sci USA 2009;106: Sijmons PC, Dekker BM, Schrammeijer B, Verwoerd TC, van den Elzen PJM, Hoekema 6099–104. AA. Production of correctly processed human serum albumin in transgenic plants. Paul M, Ma JK. Plant-made immunogens and effective delivery strategies. Expert Rev Biotechnology (NY) 1990;8:217–21. Vaccin 2010;9:821–33. Singh AK, Verma SS. Plastid transformation in eggplant (Solanum melongena L.). Peebles CAM, Gibson SI, Shanks JV, San KY. Characterization of an ethanol-inducible Transgenic Res 2010;19:113–9. promoter system in Catharanthus roseus hairy roots. Biotechnol Prog 2007;23: Sojikul P, Buehner N, Mason HS. A plant signal peptide hepatitis B surface antigen fusion 1258–60. protein with enhanced stability and immunogenicity expressed in plant cells. Proc Philip R, Darnowski DW, Maughan PJ, Vodkin LO. Processing and localization of bovine Natl Acad Sci USA 2003;100:2209–14. b-casein expressed in transgenic soybean seeds under control of a soybean lectin Sourrouille C, Marquet-Blouin E, D'Aoust MA, Kiefer-Meyer M-C, Seveno M, Pagny- expression cassette. Plant Sci 2001;161:323–35. Salehabadi S, et al. Down-regulated expression of plant-specific glycoepitopes in Plasson C, Michel R, Lienard D, Saint-Jore-Dupas C, Sourrouille C, de March GG, et al. alfalfa. Plant Biotechnol J 2008;6:702–21. Production of recombinant proteins in suspension-cultured plant cells. Meth Mol Sparrow PA, Irwin JA, Dale PJ, Twyman RM, Ma JK. Pharma-Planta: road testing the Biol 2009;483:145–61. developing regulatory guidelines for plant-made pharmaceuticals. Transgenic Res Pogue GP, Vojdani F, Palmer KE, Hiatt E, Hume S, Phelps J, et al. Production of 2007;16:147–61. pharmaceutical-grade recombinant aprotinin and a monoclonal antibody product Spök A. Molecular farming on the rise—GMO regulators still walking a tightrope. Trends using plant-based transient expression systems. Plant Biotechnol J 2010;8:638–54. Biotechnol 2007;25:74–82. Porta C, Lomonossoff GP. Viruses as vectors for the expression of foreign sequences in Spök A, Twyman RM, Fischer R, Ma JK, Sparrow PA. Evolution of a regulatory framework plants. Biotechnol Genet Eng Rev 2002;19:245–91. for pharmaceuticals derived from genetically modified plants. Trends Biotechnol Pujol M, Ramírez NI, Ayala M, Gavilondo JV, Valdés R, Rodríguez M, et al. An integral 2008;26:506–17. approach towards a practical application for a plant-made monoclonal antibody in Stefanova G, Vlahova M, Atanassov A. Production of recombinant human lactoferrin vaccine purification. Vaccine 2005;23:1833–7. from transgenic plants. Biol Plant 2008;52:423–8. Rademacher T, Sack M, Arcalis E, Stadlmann J, Balzer S, Altmann F, et al. Recombinant Sticklen MB. Plant genetic engineering for biofuel production: towards affordable antibody 2G12 produced in maize endosperm efficiently neutralizes HIV-1 and cellulosic ethanol. Nat Rev Gen 2008;9:433–43. contains predominantly single-GlcNAc N-glycans. Plant Biotechnol J 2008;6:189–201. Stoger E, Sack M, Fischer R, Christou P. Plantibodies: applications, advantages and Ramessar K, Sabalza M, Capell T, Christou P. Maize plants: an ideal production platform bottlenecks. Curr Opin Biotechnol 2002;13:161–6. for effective and safe molecular pharming. Plant Sci 2008a;174:409–19. Stoger E, Ma JKC, Fischer R, Christou P. Sowing the seeds of success: pharmaceutical Ramessar K, Rademacher T, Sack M, Stadlmann J, Platis D, Stiegler G, et al. Cost-effective proteins from plants. Curr Opin Biotechnol 2005a;16:167–73. production of a vaginal protein microbicide to prevent HIV transmission. Proc Natl Stoger E, Sack M, Nicholson L, Fischer R, Christou P. Recent progress in plantibody Acad Sci USA 2008b;105:3727–32. technology. Curr Pharm Des 2005b;11:2439–57. Reddy VS, Sadhu L, Selvapandiyan A, Raman R, Giovanni F, Shukla V, et al. Analysis of Streatfield SJ, Howard JA. Plant-based vaccines. Int J Parasitol 2003;33:479–93. chloroplast transformed tobacco plants with cry1Ia5 under rice psbA transcrip- Streatfield SJ. Mucosal immunization using recombinant plant-based oral vaccines. tional elements reveal high level expression of Bt toxin without imposing yield Methods 2006;38:150–7. penalty and stable inheritance of transplastome. Mol Breed 2002;9:259–69. Streatfield SJ. Approaches to achieve high-level heterologous protein production in Regnard GL, Halley-Stott RP, Tanzer FL, Hitzeroth II, Rybicki EP. High level protein plants. Plant Biotechnol J 2007;5:2-15. expression in plants through the use of a novel autonomously replicating Sugio T, Matsuura H, Matsui T, Matsunaga M, Nosho T, Kanaya S, et al. Effect of the geminivirus shuttle vector. Plant Biotechnol J 2010;8:38–46. sequence context of the AUG initiation codon on the rate of translation in Richter LJ, Thanavala Y, Arntzen CJ, Mason HS. Production of hepatitis B surface antigen dicotyledonous and monocotyledonous plant cells. J Biosci Bioeng 2010;109: in transgenic plants for oral immunization. Nat Biotechnol 2000;18:1167–71. 170–3. Rosales-Mendoza S, Alpuche-Solıs AG, Soria-Guerra RE, Moreno-Fierros L, Martínez- Svab Z, Maliga P. Exceptional transmission of plastids and mitochondria from the González L, Herrera-Díaz A, et al. Expression of an Escherichia coli antigenic fusion transplastomic pollen parent and its impact on transgene containment. Proc Natl protein comprising the heat labile toxin B subunit and the heat stable toxin, and its Acad Sci USA 2007;104:7003–8. assembly as a functional oligomer in transplastomic tobacco plants. Plant J 2009;57: Tacket CO. Plant-derived vaccines against diarrheal diseases. Vaccine 2005;23:1866–9. 45–54. Takagi H, Hiroi T, Yang L, Tada Y, Yuki Y, Takamura K, et al. A rice-based edible vaccine Rosales-Mendoza S, Soria-Guerra RE, Moreno-Fierros L, Alpuche-Solís ÁG, Martínez- expressing multiple T cell epitopes induces oral tolerance for inhibition of Th2- González L, Korban SS. Expression of an immunogenic F1-V fusion protein in lettuce as mediated IgE responses. Proc Natl Acad Sci USA 2005;102:17525–30. a plant-based vaccine against plague. Planta 2010, doi:10.1007/s00425-010-1176-z. Terpe K. Overview of tag protein fusions: from molecular and biochemical Rotino GL, Acciarri N, Sabatini E, Mennella G, Lo Scalzo R, Maestrelli A, et al. Open field fundamentals to commercial systems. Appl Microbiol Biotechnol 2003;60:523–33. trial of genetically modified parthenocarpic tomato: seedlessness and fruit quality. Thanavala Y, Mahoney M, Pal S, Scott A, Richter L, Natarajan N, et al. Immunogenicity in BMC Biotechnol 2005;5. humans of an ediblevaccine for hepatitis B. Proc Natl Acad Sci USA 2005;102: Russell DA, Spatola LA, Dian T, Paradkar VM, Dufield DR, Carroll JA, et al. Host limits to 3378–82. accurate human growth hormone production in multiple plant systems. Biotechnol Tiwari S, Verma PC, Singh PK, Tuli R. Plants as bioreactors for the production of vaccine Bioeng 2005;89:775–82. antigens. Biotechnol Adv 2009;27:449–67. Rybicki EP. Plant-produced vaccines: promise and reality. Drug Discov Today 2009;14: Torrent M, Llompart B, Lasserre-Ramassamy S, Llop-Tous I, Bastida M, Marzabal P, 16–24. et al. Eukaryotic protein production in designed storage organelles. BMC Biol Rybicki EP. Plant-made vaccines for humans and animals. Plant Biotechnol J 2010;8: 2009a;7:5. 620–37. Torrent M, Llop-Tous I, Ludevid MD. Protein body induction: a new tool to produce and Sadhu L, Reddy VS. Chloroplast expression of His-tagged GUS-fusions: a general recover recombinant proteins in plants. Meth Mol Biol 2009b;483:193–208. strategy to overproduce and purify foreign proteins using transplastomic plants as Tran M, Zhou B, Pettersson PL, Gonzalez MJ, Mayfield SP. Synthesis and assembly of a bioreactors. Mol Breed 2003;11:49–58. full-length human monoclonal antibody in algal chloroplasts. Biotechnol Bioeng Sandhu S, James VA, Quesenberry KH, Altpeter F. Risk assessment of transgenic 2009;104:663–73. apomictic tetraploid bahiagrass, cytogenetics, breeding behavior and performance Tregoning JS, Clare S, Bowe F, Edwards L, Fairweather N, Qazi O, et al. Protection against of intra-specific hybrids. Theor Appl Genet 2009;119:1-13. tetanus toxin using a plant-based vaccine. Eur J Immunol 2005;35:1320–6. Santi L, Giritch A, Roy CJ, Marillonnet S, Klimyuk V, Gleba Y, et al. Protection conferred Tremblay R, Wang D, Jevnikar AM, Ma S. Tobacco, a highly efficient green bioreactor for by recombinant Yersinia pestis antigens produced by a rapid and highly scalable production of therapeutic proteins. Biotechnol Adv 2010;28:214–21. plant expression system. Proc Natl Acad Sci USA 2006;103:861–6. US Department of Agriculture. BRS factsheet. . Available from:USDA-APHIS; 2006. http: Satyavathi VV, Prasad V, Khandelwal A, Shaila MS, Sita GL. Expression of hemagglutinin //www.aphis.usda.gov/publications/biotechnology/content/printable_version/ protein of rinderpest virus in transgenic pigeon pea [Cajanus cajan (L.) Millsp.] BRS_FS_pharmaceutical_02-06.pdf. accessed 20/8/09. plants. Plant Cell Rep 2003;21:651–8. USDA/APHIS. Draft guidance for APHIS permits for field testing or movement of Schaefer DG. A new moss genetics: targeted mutagenesis in Physcomitrella patens. Annu organisms with pharmaceutical or industrial intent; 2006. http://www.aphis.usda. Rev Plant Biol 2002;53:477–501. gov/brs/pdf/Pharma_Guidance.pdf. accessed 20/8/09. 222 O.O. Obembe et al. / Biotechnology Advances 29 (2011) 210–222

Varsani A, Williamson AL, Rose RC, Jaffer M, Rybicki EP. Expression of human Wisner R. The economics of pharmaceutical crops: potential benefits and risks for papillomavirus type 16 major capsid protein in transgenic Nicotiana tabacum cv. farmers and rural communities, Union of Concerned Scientists. Sep. Available from: Xanthi. Arch Virol 2003;148:1771–86. http://www.ucsusa.org/food_and_environment/genetic_engineering/economics- Varsani A, Williamson A-L, Stewart D, Rybicki EP. Transient expression of human of-pharmaceutical-crops.html. accessed 20/8/09. papillomavirus type 16L1 protein in Nicotiana benthamiana using an infectious Woodard SL, Mayor JM, Bailey MR, Barker DK, Love RT, Lane JR, et al. Maize (Zea mays)- tobamovirus vector. Virus Res 2006;120:91–6. derived bovine trypsin: characterization of the first large-scale, commercial protein Vézina L-P, Faye L, Lerouge P, D'Aoust M-A, Marquet-Blouin E, Burel C, et al. Transient co- product from transgenic plants. Biotechnol Appl Biochem 2003;38:123–30. expression for fast and high-yield production of antibodies with human-like N-glycans Xu B, Copolla M, Herr JC, Timko MP. Expression of a recombinant human sperm- in plants. Plant Biotechnol J 2009;7:442–55. agglutinating mini-antibody in tobacco (Nicotiana tabacum L). Soc Reprod Fertil Villani ME, Morgun B, Brunetti P, Marusic C, Lombardi R, Pisoni I, et al. Plant pharming Suppl 2007;63:465–77. of a full-sized, tumour-targeting antibody using different expression strategies. Yamada Y, Nishizawa K, Yokoo M, Zhao H, Onishi K, Teraishi M, et al. Anti-hypertensive Plant Biotechnol J 2008;6:59–72. activity of genetically modified soybean seeds accumulating novokinin. Peptides Voinnet O, Rivas S, Mestre P, Baulcombe D. An enhanced transient expression system in 2008;29:331–7. plants based on suppression of gene silencing by the p19 protein of tomato bushy Yang D, Wu L, Hwang YS, Chen L, Huang N. Expression of the REB transcriptional activator stunt virus. Plant J 2003;33:949–56. in rice grains improves the yield of recombinant proteins whose genes are controlled Walmsley AM, Arntzen CJ. Plant cell factories and mucosal vaccines. Curr Opin by a Reb-responsive promoter. Proc Natl Acad Sci USA 2001;98:11438–43. Biotechnol 2003;14:145–50. Yang D, Guo F, Liu B, Huang N, Watkins SC. Expression and localization of human Weise A, Altmann F, Rodríguez-Franco M, Sjoberg ER, Bäumer W, Launhardt H, et al. High- lysozyme in the endosperm of transgenic rice. Planta 2003;216:597–603. level expression of secreted complex glycosylate recombinant human erythropoietin Yano M, Hirai T, Kato K, Hiwasa-Tanase K, Fukuda N, Ezura H. Tomato is a suitable in the Physcomitrella D-fuc-t D-xyl-t mutant. Plant Biotechnol J 2007;5:389–401. material for producing recombinant miraculin protein in genetically stable manner. Wilde CD, Peeters K, Jacobs A, Peck I, Depicker A. Expression of antibodies and Fab Plant Sci 2010;178:469–73. fragments in transgenic potato plants: a case study for bulk production in crop Zeitlin L, Pauly M, Whaley KJ. Second-generation HIV microbicides: continued plants. Mol Breed 2002;9:271–82. development of griffithsin. Proc Natl Acad Sci USA 2009;106:6029–30. Wirth S, Calamante G, Mentaberry A, Bussmann L, Lattanzi M, Barañao L, et al. Zhu Z, Hughes KW, Huang L, Sun B, Liu C, Li Y. Expression of human alpha-interferon Expression of active human epidermal growth factor (hEGF) in tobacco plants by cDNA in transgenic rice plants. Plant Cell Tissue Organ Cult 2004;36:197–204. integrative and non-integrative systems. Mol Breed 2004;13:23–35.